Journal: Science signaling

Article Title: Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

doi: 10.1126/scisignal.aad3812

Figure Lengend Snippet: Fig. 1. VEGF-A165, a ligand-blocking anti-NRP1, and a tetrameric CendR peptide induce NRP1 accumula- tion at endothelial cell–cell contacts. (A to D) Epifluorescence microscope images of permeabilized HUVEC monolayers. (A) Cells were stimulated with VEGF-A165 and stained with an antibody against NRP1 (red). (B) After incubation with the ligand-blocking anti-NRP1 antibody (anti-NRP1) (lower panel) or its cor- responding control sheep IgG (upper panel), cells were stimulated with VEGF-A165. HUVECs were stained with an antibody specific for NRP1 (red) and secondary anti-sheep antibody (green). Accumulation of NRP1 at cell-cell contacts was observed in the absence of VEGF-A165 (white arrows). (C) Cells were stimulated with anti-NRP1 and stained with secondary anti-sheep antibody (green). (D) Cells were stimulated with NA-RPARPAR peptide and stained with an antibody specific for NRP1 (red). Nuclei were stained with Hoechst (blue). Images in (A) to (D) are representative of n > 3 independent experiments. Scale bars, 20 mm.

Article Snippet: The ligand-blocking sheep anti-human NRP1 and goat anti-rat NRP1 antibodies were purchased from R&D Systems.

Techniques: Blocking Assay, Microscopy, Staining, Incubation, Control

Journal: Science signaling

Article Title: Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

doi: 10.1126/scisignal.aad3812

Figure Lengend Snippet: Fig. 2. VEGF-A165, NA-RPARPAR, and anti-NRP1 induce NRP1 junctional localization and endothelial leakage in vitro and in vivo. (A) Confocal microscopy images of permeabilized HUVEC monolayers stimu- lated with VEGF-A165, NA-RPARPAR, and anti-NRP1 and stained with an antibody against VE-cadherin (green). NRP1 (red) was stained with an antibody against NRP1 (upper and middle panels) or directly with the secondary anti-sheep antibody (lower panel). Nuclei were stained with Hoechst (blue). White arrows indicate colocalization between NRP1 and VE-cadherin. Images are representative of four independent experiments. Scale bar, 20 mm. (B) HUVEC monolayers were seeded on top of Transwell filters and stimulated with PBS, VEGF-A165 (VEGF), NA-RPARPAR (NA-R), and anti-NRP1 (Ab). Leakage of FITC- dextran 70 kD from the upper to the lower well was measured by comparing fluorescence values at 520 nm (mean ± SEM; n = 9 independent experiments; the median from three to six replicates per independent experiment was used for statistical analyses; Friedman test followed by Dunn’s multiple com- parison post hoc test; *P < 0.05 and **P < 0.01). (C) Wild-type mice were systemically injected with Evans Blue and then with PBS, VEGF-A165, NA-RPARPAR, anti-NRP1 antibody, and their respective controls. The extravasated dye concentration was measured at 620 nm, and results were expressed as a ratio between the tested substance and its control (mean; n = 10 to 15 mice per treatment; Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test; **P < 0.01 and ***P < 0.001).

Article Snippet: The ligand-blocking sheep anti-human NRP1 and goat anti-rat NRP1 antibodies were purchased from R&D Systems.

Techniques: In Vitro, In Vivo, Confocal Microscopy, Staining, Fluorescence, Injection, Concentration Assay, Control, Comparison

Journal: Science signaling

Article Title: Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

doi: 10.1126/scisignal.aad3812

Figure Lengend Snippet: Fig. 3. Unlike VEGF-A165, NA-RPARPAR and anti-NRP1 do not activate VEGFR-2, Akt, p38, ERK, or FAK. (A to C) HUVEC monolayers were stimu- lated with VEGF-A165, NA-RPARPAR, or anti-NRP1 antibody. (A) Cell ly- sates were immunoprecipitated with an antibody against VEGFR-2 and blotted with anti–VEGFR-2 and anti-phosphotyrosine (pY) antibodies. The corresponding total lysates were blotted for VEGFR-2 (n = 4 independent experiments). (B and C) Blotting was performed on total lysates with anti- bodies against pAkt, pERK1/2, p-p38 (n = 3 independent experiments) (B),

Article Snippet: The ligand-blocking sheep anti-human NRP1 and goat anti-rat NRP1 antibodies were purchased from R&D Systems.

Techniques: Immunoprecipitation

Journal: Science signaling

Article Title: Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

doi: 10.1126/scisignal.aad3812

Figure Lengend Snippet: Fig. 4. VEGF-A165, NA-RPARPAR, and anti-NRP1 induce NRP1 relocalization and vascular leakage inde- pendently of VEGFR-2 activation. (A and B) HUVEC monolayers were treated with PTK/ZK or dimethyl sulfoxide (DMSO) before stimulation. ct, control. (A) Cell lysates were immunoprecipitated (IP) with VEGFR-2 antibody. Immunoprecipitates were blotted for VEGFR-2 and phosphotyrosine, and corresponding total lysates for VEGFR-2 (R2). Representative scans of five experiments. (B) HUVEC mono- layers were stained for NRP1 (red), nuclei were stained with Hoechst (blue), and cells were imaged with an epifluorescence microscope. Representative images from three experiments. Scale bar, 25 mm. (C and D) HUVECs were transfected with noncoding (NC) small interfering RNA (siRNA) or siRNAs coding for VEGFR-2 (KDR) (siRNA 1 and siRNA 2). (C) KDR mRNA relative expression was quantified by quantita- tive real-time polymerase chain reaction (qRT-PCR) (mean ± SEM; n = 3 independent experiments). (D) Epifluorescence images of transfected HUVECs in (C), stimulated with VEGF-A165, NA-RPARPAR, or anti- NRP1. Cells were stained for NRP1 (red), and nuclei were stained with Hoechst (blue). Representative images of three experiments. Scale bar, 20 mm. (E to H) Mice were injected intraperitoneally with DMSO or PTK/ZK before systemic treatment with Evans Blue. Leakage was induced with VEGF-A165 (E), NA-RPARPAR (F), anti-NRP1 (G), or VEGF-A121 (H), and results were expressed as a ratio between the tested substance and its respective control (mean; n = 10 to 20 mice per condition; Mann-Whitney test).

Article Snippet: The ligand-blocking sheep anti-human NRP1 and goat anti-rat NRP1 antibodies were purchased from R&D Systems.

Techniques: Activation Assay, Control, Immunoprecipitation, Staining, Microscopy, Transfection, Small Interfering RNA, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Injection, MANN-WHITNEY

Journal: Science signaling

Article Title: Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

doi: 10.1126/scisignal.aad3812

Figure Lengend Snippet: Fig. 5. The NRP1 cytoplasmic domain mediates vascular leakage. (A and B) HUVECs overexpressing GFP, full-length NRP1, or NRP1 deprived from the cytoplasmic domain (NRP1DC) were stimulated with VEGF-A165. (A) Cell lysates were immunoprecipitated with VEGFR-2 antibody. Immunopre- cipitates were blotted for VEGFR-2 and phosphotyrosine, and total lysates were blotted for NRP1 and actin (n = 3 independent experiments). (B) HUVECs overexpressing GFP, NRP1, or NRP1DC were stained with anti- NRP1 (red). Nuclei were stained with Hoechst (blue). Representative images of three independent experiments. Scale bar, 20 mm. (C to E) Wild-type mice (NRP1cyto+/+) and mice expressing cytoplasmatically trun-

Article Snippet: The ligand-blocking sheep anti-human NRP1 and goat anti-rat NRP1 antibodies were purchased from R&D Systems.

Techniques: Immunoprecipitation, Staining, Expressing

Journal: Journal of the American Society of Nephrology

Article Title: Chordin-like 1 and Twisted Gastrulation 1 Regulate BMP Signaling following Kidney Injury

doi: 10.1681/asn.2008070768

Figure Lengend Snippet: Figure 4. CHRDL1 functions as a general BMP-signaling amplifier but specifically antagonizes BMP7 in the presence of TWSG1. (A) P19 embryonal carcinoma cells transfected with the BMP transcriptional reporter pBRE-Luc were incubated overnight with BMP4 (5 ng/ml) or BMP7 (10 ng/ml) and increasing amounts of CHRDL1 (50 to 400 ng/ml) or Chordin (100 to 800 ng/ml). Both CHRDL1 and Chordin amplify BMP4 signaling; however, in contrast to Chordin, which antagonizes BMP7 signaling, CHRDL1 amplifies BMP7 signaling in a dosage-responsive manner. (B) In the presence of TWSG1 (100 to 400 ng/ml), CHRDL1 (200 ng/ml) continues to act as an amplifier of BMP4 signaling unlike Chordin (400 ng/ml), which becomes a potent BMP4 antagonist. In contrast, CHRDL1 amplification of BMP7 signaling is converted to antagonism by addition of TWSG1 in a dosage-dependent manner. (C) In the P19 pBRE-luc reporter assay, the addition of TWSG1, CHRDL1, or Chordin (400 ng/ml) alone does not affect transcriptional activation. (D) CHRDL1 binds BMP7 and TWSG1 only in a trimolecular complex. A co-immunoprecipitation experiment in which CHRDL1 (500 ng/ml) is incubated in the presence of TWSG1 (500 ng/ml) and/or BMP7 (250 ng/ml) demonstrates that CHRDL1 binds TWSG1 and BMP7 only when all three proteins are present. No evidence of binding is detected when TWSG1 or BMP7 alone is incubated with CHRDL1. Recombinant proteins were immunoprecipitated with a goat polyclonal antibody against CHRDL1, and blots of goat IgG serve as loading controls. The last panel, a CHRDL1 immunoblot of an identical immunoprecipitation substituting an irrelevant goat antibody, demonstrates the absence of nonspecific binding by CHRDL1 to either the goat antibody or Protein G beads used in the experiment. The first lane of all blots contains recombinant protein as a positive control. (E) TWSG1 is strongly expressed in tubule epithelia of the mouse and human kidney. (Top) Strong immunohistochemical staining for TWSG1 in tubule epithelia of adult mouse kidney but weak staining in the glomerulus (G) and interstitial cell population (inset). (Right) Negative control using an antibody of the same species specific for macrophage. (Bottom) Immunofluorescent micro- graphs of an adult human kidney showing TWSG1 expression (red) in both proximal tubules (PT), marked green with Lotus lectin, and other tubules. As in the mouse, glomerular expression of TWSG1 is much weaker than seen in tubular epithelia. (Right) Negative control for TWSG1.

Article Snippet: BMP7 was detected with mouse anti- BMP7 (R&D Systems) and a sheep anti-mouse HRP conjugate (Am- ersham).

Techniques: Transfection, Incubation, Amplification, Reporter Assay, Activation Assay, Immunoprecipitation, Binding Assay, Recombinant, Western Blot, Positive Control, Immunohistochemical staining, Staining, Negative Control, Expressing

Journal: Journal of the American Society of Nephrology

Article Title: Chordin-like 1 and Twisted Gastrulation 1 Regulate BMP Signaling following Kidney Injury

doi: 10.1681/asn.2008070768

Figure Lengend Snippet: Figure 5. CHRDL1 reduces BMP7-stimulated Smad activation and ID gene expression in the presence of TWSG1 but has no effect on BMP4 signaling. (A and B) In the presence of TWSG1, CHRDL1 inhibits Smad phosphorylation by BMP7 but not by BMP4. P19 cells were serum starved for 2 h and then incubated with BMPs (10 ng/ml) and antagonists for 2 h before lysis and Western blotting. Blots were probed for phosphorylated Smads 1, 5, and 8 and -tubulin. In lanes 3, 4, and 5, Noggin (200 ng/ml), TWSG1, and CHRDL1 alone (400 ng/ml) were added. In lanes 6 through 9 CHRDL1 (400 ng/ml) was added together with increasing concentrations of TWSG1 (50, 100, 200, and 400 ng/ml). BMPs were incubated at 37 C for 1 h with or without antagonists before application. Note that the arrow points to the band corresponding to pSmad1/5/8. The top band (arrowhead) is a contaminating band specific to P19 lysates and is not present in lysates from MDCK cells (compare C and D). (C and D) The effects of CHRDL1 and TWSG1 on BMP signaling can be reproduced in the MDCK kidney cell line. (E) RT-PCR showing expression of ID1, 2, and 3 genes in HK-2 cells stimulated with BMP4 or 7 (25 ng/ml) for 6 h in the presence of varying concentrations (100 to 400 ng/ml) of CHRDL1 and/or TWSG1. ID expression by HK-2 cells in response to BMP4 is unaffected by the presence of TWSG1. In cells incubated with BMP7, the addition of TWSG1 reduces ID gene expression.

Article Snippet: BMP7 was detected with mouse anti- BMP7 (R&D Systems) and a sheep anti-mouse HRP conjugate (Am- ersham).

Techniques: Activation Assay, Gene Expression, Phospho-proteomics, Incubation, Lysis, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Journal of the American Society of Nephrology

Article Title: Chordin-like 1 and Twisted Gastrulation 1 Regulate BMP Signaling following Kidney Injury

doi: 10.1681/asn.2008070768

Figure Lengend Snippet: Figure 6. Overexpression of mouse Chrdl1 in the collecting duct substan- tially reduces BMP signaling in vivo. (A) Diagram of mouse Chrdl1 transgene construct driven by a collecting duct–specific enhancer element from intron 1 of the Bmp7 gene; quantitative PCR assay shows Chrdl1 overexpression in kidneys of two embryonic day 17.5 transgenic embryos (150 and 151) com- pared with wild-type. (B through D) Immunolocalization of phosphorylated Smads (red) in the embryonic day 17.5 wild-type kidney (B), and kidneys from transgenic embryos 150 and 151 (C and D) show that BMP signaling is substantially reduced by expression of the Chrdl1 transgene. Collecting ducts (CD) were localized by staining with the lectin Dolichos Biflorus Agglu- tinin (green), and nuclei were counterstained with DAPI (blue).

Article Snippet: BMP7 was detected with mouse anti- BMP7 (R&D Systems) and a sheep anti-mouse HRP conjugate (Am- ersham).

Techniques: Over Expression, In Vivo, Construct, Real-time Polymerase Chain Reaction, Transgenic Assay, Expressing, Staining